DESCRIPTION: This is a proposal to investigate the role of reactive nitrogen species in skin tumor promotion in mice. Considerable recent evidence indicates that both reactive nitrogen and oxygen species are formed in skin treated with tumor promoter tetradecanoylphorbol acetate (TPA) in a multistage carcinogenesis (DMBA/TPA) model and in skin treated chronically with the complete carcinogen DMBA. The hypothesis to be investigated in this project is that reactive nitrogen intermediates produced by inflammatory leukocytes present in the dermis and the dermal/epidermal junction cause oxidative DNA damage that leads to mutations and affects clonal expansion of initiated cell populations through the selective induction of apoptosis. This hypothesis will be addressed through three specific aims. Studies in aim 1 will characterize changes in the expression of genes for inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) as well as the induction of apoptosis in epidermis of mice subjected to a DMBA/TPA protocol or multiple treatments with DMBA alone. Studies in Aim 2 will investigate the effects of modulation of NO production with the NOS inhibitor monomethyl-L-arginine monoacetate (L-NMMA) or the NO-releasing compound DETA/NO in the DMBA/TPA or DMBA-only protocols. Parameters to be investigated include NOS gene expression, vascular permeability, skin thickness, vascular endothelial growth factor and vascular permeability factor induction, NO and peroxynitrite production, leukocyte infiltration, apoptosis, and oxidative DNA damage. Studies in aim 3 will attempt to determine the nature of the reactive nitrogen species involved in mutagenesis and the mutations they produce in an in vitro co-culture system employing AS52 cells exposed to oxidants or incubated in co-culture with peripheral leukocytes isolated from DMBA/TPA or DMBA treated mice. Mutations in the gpt gene of the AS52 cells will be characterized.